Search

Cyclic nucleotide-gated (CNG) channels play an essential role in the sensory transduction of the olfactory and visual system. Native olfactory CNG channels are heterotetramers composed of three different subunits: two CNGA2, one CNGA4 and one CNGB1b. It is well established that the presence of the CNGA4 and CNGB1b subunit in heterotetrameric channels leads to an increased apparent affinity for cyclic nucleotides. We have recently shown that the CNGA4 subunit binds cGMP and actively participates to channel activation. Nevertheless, the functional contribution of the CNGB1b subunit is still unclear. By combining mutagenesis and confocal patch-clamp fluorometry we characterized the role of the CNGB1b subunit during channel activation. We show that in contrast to the CNGA4 subunit, that needs only neighbouring CNGA2 subunits for ligand binding, the CNGB1b needs both, CNGA2 and CNGA4. When coexpressing CNGB1b with disabled CNGA2 and CNGA4, channel activation induced by the CNGB1b subunit alone was only weak compared to CNGA4 coexpressed with disabled CNGA2. The apparent affinity (EC50) of the CNGB1b subunit under the same conditions is 3.2 µM. This is twofold higher than that determined for CNGA4 and 15 fold higher than that of one CNGA2. The binding of cAMP and cGMP to the CNGB1b subunit turned out to be non-selective. Moreover, the kinetics of ligand binding and unbinding as well as channel activation and deactivation for each of the three different subunits in the respective environment of disabled subunits, substantiate the role of the CNGB1b and CNGA4 in speeding up channel closure. In conclusion, we show that olfactory CNG channels are activated by ligand binding to all four subunits, but the contribution of the CNGB1b to channel activation is only modest in comparison with that of the other subunits.

HCN pacemaker channels play an important role in generating and regulating rhythmicity of special neurons and cardiac cells. They are activated by hyperpolarizing voltages and modulated by the binding of cyclic nucleotides. Four isoforms, HCN1-HCN4, have been identified. HCN2 and HCN4, expressed in cardiac sinoatrial node and ventricular cells, build functional homotetrameric and heterotetrameric channels in various heterologous cell systems.Heterotetrameric HCN2/4 channels in Xenopus oocytes show two changes compared to each of the homotetramers: the voltage of half maximum activation is shifted to more depolarized voltages and activation kinetics are faster (Zhang et al., Biochim Biophys Acta, 2009). However, little is known about the ligand dependence of these channels. Herein, we studied the differences in activation gating of HCN2/4 channels in comparison to the respective homotetramers, thereby focusing on the effect of cAMP. We monitored activation under both steady-state and non steady-state conditions in the presence and absence of cAMP, as well as the ligand-dependent activation kinetics after ligand jumps.We found (1) that in HCN2/4 the apparent affinity for cAMP was between that of the two homotetramers, whereas the Hill coefficient was lowest, (2) that cAMP accelerates voltage-induced activation in HCN2/4 only slightly (factor ∼2), resembling HCN4, whereas in HCN2 it accelerates activation in a voltage-dependent manner by a factor of up to ∼12, (3) that the activation kinetics following a cAMP jump to channels pre-activated by voltage was fastest in HCN2/4, whereas the increase of current amplitude by a concentration jump was similar to homotetramers. Our results confirm that coinjection of the HCN2 and HCN4 isoforms in Xenopus oocytes leads to heterotetrameric channels. They suggest that in native heart cells the formation of heterotetramers leads to pacemaker channels with specific characteristics, thereby fine-tuning the process of pacemaking.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are voltage-gated tetrameric cation channels that generate electrical rhythmicity in neurons and cardiomyocytes. Activation can be enhanced by the binding of adenosine-3′,5′-cyclic monophosphate (cAMP) to an intracellular cyclic nucleotide binding domain. Based on previously determined rate constants for a complex Markovian model describing the gating of homotetrameric HCN2 channels, we analyzed probability fluxes within this model, including unidirectional probability fluxes and the probability flux along transition paths. The time-dependent probability fluxes quantify the contributions of all 13 transitions of the model to channel activation. The binding of the first, third and fourth ligand evoked robust channel opening whereas the binding of the second ligand obstructed channel opening similar to the empty channel. Analysis of the net probability fluxes in terms of the transition path theory revealed pronounced hysteresis for channel activation and deactivation. These results provide quantitative insight into the complex interaction of the four structurally equal subunits, leading to non-equality in their function., Author Summary The activation of a receptor protein by a small molecule (ligand) can be quantified by Markovian models. These models, which are widely used in natural sciences, consist of distinct states and transitions between them. In nature receptor proteins are often formed by the assembly of more than one subunit and each subunit can bind a ligand on its own. In such a multimeric receptor protein the translation of the ligand binding into receptor activation is more complex because the subunits interact. This usually limits the application of Markovian models. HCN2 pacemaker channels are tetrameric ion channels that mediate electrical rhythmicity in multiple brain and peripheral neurons and in specialized heart cells. The channels are modulated by cAMP binding to each subunit. We were recently successful to quantify ligand-induced activation for these channels by a complex Markovian model and a full set of rate constants. Herein we applied the transition path theory to further analyze the identified Markovian model, and we quantified time-dependent probability fluxes within the model. Our results provide unprecedented insight into the complex interaction of the four structurally equal subunits of a presumably fourfold symmetric channel that leads to pronounced non-equality of the subunit function.

The term ‘cooperativity’ is generally used to functionally characterize enzymes and receptors with multiple binding sites when the affinity at one binding site influences the affinity at the other binding site(s). The affinity increase for the primary ligand is a simple energetic consequence of its action because the energy used for a confomational change must be provided by the binding itself. The affinity change in the other binding sites is a speciality of the multimeric protein, determining its characteristic properties. An affinity increase of the subsequent binging sites is usually termed ‘positive cooperativity’ and an affinity decrease ‘negative cooperativity’. Positive cooperativity has been often described by the Monod-Wyman-Changeux model, assuming a constant allosteric factor for the successive affinity increase for each binding step. Using confocal patch-clamp fluorometry and a fluorescence-labelled cAMP, we recently determined for HCN2 channels the rate constants in a Markovian model containing four sequential binding steps, in both the closed and the open channel, and five closed open isomerizations. The equilibrium association constants of the binding steps, i.e. the affinity of the binding sites, changed with the sequence 1.5×106, 9.0×106, 1.2×104, 2.6×106 (M−1), resulting in a sequence of cooperativity of ‘positive-negative-positive’. However, cooperativity can also be related to the rates of binding and unbinding, i.e. to physical processes proceeding in time. If considering, for example, the binding rates in the closed channel, the sequence is 5.4×106, 8.4×105, 9.9×104, 2.2×107 (M−1s−1), resulting in a sequence of cooperativity of ‘negative-negative-positive’. In the open channel this sequence is similar. As a conclusion we propose to distinguish between cooperativity related to the affinities of the binding sites from cooperativity related to the rates of binding or unbinding at the binding sites, providing more precise information about the underlying physical processes.

Restoration of the blood flow in the ischemic myocardium can result in a lethal reperfusion injury. Reactive oxygen species (ROS) that are formed in the presence of oxygen are believed to play an important role in the post-ischemic injury. Reperfusion of the myocardium is always accompanied by the reoxygenation. Therefore the effect of reoxygenation alone is unknown. Here, using oxygen clamp in on-chip picochambers we exposed single resting cardiomyocytes to near anoxia (pO2 < 0.1 mm Hg) and subsequently to normoxia. As cardiomyocytes were trapped in picochambers no reperfusion occurred. This allowed us to study the effects of reoxygenation independent of reperfusion. The mitochondrial membrane potential (ΔΨ) was measured in cardiomyocytes simultaneously with IKATP, providing a measure for the cytosolic ATP. We show that the reoxygenation of ischemic cardiomyocytes alone is insufficient to induce a high ATP turnover rate but that the reperfusion is required for it. Upon reoxygenation to normoxia ΔΨ transiently (6.0 ± 0.7 s) undershooted the pre-ischemic level. The post-anoxic undershoot of ΔΨ was associated with the increased rate of ATP synthesis, indicating that it was not due to substrate limitation of the F0F1-ATPase. It persisted in cells with stimulated (FCCP; duration 7 ± 1 s) and inhibited (rotenone; 6 ± 2 s) respiration. However, in the presence of antimycin, which is known to promote the formation of ROS, the post-anoxic undershoot of ΔΨ was prolonged to 28 ± 3 s. ATP synthesis was also promoted. We propose that the ROS burst after reoxygenation from near anoxia by donating electrons to complex IV essentially contributes to a transient stimulation of respiration and promote the post-anoxic recovery of cellular ATP.

Two-pore domain (K2P) K+ channels represent a large family of ion channels that are major regulators of cellular excitability in the body and involved in a wide range of cellular mechanisms including apoptosis, vasodilatation, anaesthesia, pain, neuroprotection and temperature sensing. Many K2P channels are strongly activated by membrane depolarization, but the mechanisms underlying this voltage-dependent behaviour are unknown. Here we report that many K2P channels (e.g. TREK-1, TREK-2, TRAAK, TASK-3, TALK-2 and TRESK) are equipped with a gating machinery which directly senses the electrochemical K+ gradient and which gates the pore open when the membrane potential is positive to the K+ reversal potential. These properties couple voltage activation in K2P channels tightly to the reversal potential in distinction to classical Kv channels. We show that this sensing mechanism is located in the selectivity filter (SF), is strongly affected by the permeant ion species and operates as a check valve that is opened by outward permeation but closed by inward permeation. This gating behaviour is steeply voltage-dependent suggesting that multiple ions within the SF are moved simultaneously by the electrical field to gate the filter open. These findings highlight a mechanism of voltage-dependent gating which bypasses the need for electromechanical coupling to a separate voltage sensing module and is instead powered directly by the electrochemical gradient. This also further closes the mechanistic gap between ion channels and transporters because in both cases the electrochemical gradient is used to power a conformational change leading to either an ion translocation step in the case of a transporter or a pore gating step in K2P channels.

Cyclic nucleotide-gated (CNG) channels mediate sensory signal transduction in retinal and olfactory cells. The channels are activated by the binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus that is located at the intracellular side. The molecular events translating the ligand binding to the pore opening are still unknown. We investigated the role of the S4-S5 linker in the activation process by quantifying its interaction with other intracellular regions. To this end, we constructed chimeric channels in which the N-terminus, the S4-S5 linker, the C-linker, and the CNBD of the retinal CNGA1 subunit were systematically replaced by the respective regions of the olfactory CNGA2 subunit. Macroscopic concentration-response relations were analyzed, yielding the apparent affinity to cGMP and the Hill coefficient. The degree of functional coupling of intracellular regions in the activation gating was determined by thermodynamic double-mutant cycle analysis. We observed that all four intracellular regions, including the relatively short S4-S5 linker, are involved in controlling the apparent affinity of the channel to cGMP and, moreover, in determining the degree of cooperativity between the subunits, as derived from the Hill coefficient. The interaction energies reveal an interaction of the S4-S5 linker with both the N-terminus and the C-linker, but no interaction with the CNBD.

HCN channels are nonselective tetrameric cation channels that are activated by hyperpolarizing voltages and modulated by the ligand cAMP. They generate spontaneous rhythmic activity in heart and brain. Ligand binding to the intracellular cyclic nucleotide-binding site accelerates activation kinetics, shifts the steady-state activation to more positive voltages and increases the open probability. Though it is relatively simple to determine an apparent affinity for the ligand action, it is not so simple to determine the true ligand affinity during channel activation because, according to the principle of reciprocity, ligand binding and efficacy depend on each other, i.e. the affinity of the binding sites must increase when the channel opens. It is therefore important to determine the binding of the ligands to the channels and channel activation simultaneously.Activation of homotetrameric HCN2 channels was studied in inside-out macropatches simultaneously with ligand binding by means of patch-clamp fluorometry, using a fluorescent cAMP (fcAMP) that activates the channels in a similar manner as cAMP. With 1 μM fcAMP the binding of the ligand to the open channels exceeded that to closed channels. The slowness of the activation time course of HCN2 channels allowed us to monitor the ligand binding during the activation process. As predicted, the slow activation time course was accompanied by an increase of ligand binding. Moreover, the increase of binding was exponential whereas activation obeyed the typical sigmoidal time course. Hence, in the superimposed normalized time courses, the initial binding preceded activation whereas at later times activation preceeded binding. These results show that activation gating indeed increases the binding affinity for the ligands, quite as predicted by the principle of reciprocity, and that the gating of the first of the four subunits, which does not lead to channel opening, is associated with ligand binding.

Cyclic nucleotide-gated (CNG) channels in olfactory neurons are heterotetrameric ligand-gated cation channels that are composed of four homologue subunits (2xCNGA2, 1xCNGA4, 1xCNGB1b). Only the CNGA2 subunits form functional homotetrameric channels that can be activated in a cooperative manner by cAMP or cGMP binding to the cyclic nucleotide-binding domains (CNBD) included in each subunit. Our aim was to kinetically further dissect the molecular mechanism leading to channel activation upon ligand binding and to channel deactivation upon ligand removal.CNGA2 channels, expressed in Xenopus oocytes, were studied in excised patches by measuring simultaneously ligand binding/unbinding and activation/deactivation by means of confocal patch-clamp fluorometry under steady-state and non-steady state conditions (182 or 277 frames per second). Concentration jumps of a fluorescent cGMP derivative (Biskup et al., Nature, 446(7134): 440-3, 2007) were applied using a fast piezoelectric system. Surprisingly, the unbinding was concentration dependent while deactivation was concentration independent. The unbinding was approximately 100 times faster from fully liganded channels in comparison with the unbinding from lowly liganded channels. The obtained data were analyzed by global fits to various types of Markovian state models. The additional information of unbinding and deactivation allowed us to refine the previously determined C4L-Model (Biskup et al., Nature, 446(7134): 440-3, 2007). To account for the very fast unbinding at saturating ligand concentrations, the C4L-Model had to be expanded: When fully liganded, the channel adopts an open state which allows, upon ligand removal, a very fast unbinding of all four ligands. In contrast, from partially liganded states this fast unbinding is occluded. Our results suggest an additional pathway for rapid ligand unbinding for the fully liganded channel.

Hyperpolarization activated cyclic nucleotide-modulated (HCN) channels mediate rhythmic electrical activity in specialized brain neurons and cardiomyocytes. The channels are non-specific cation channels that are activated by hyperpolarizing voltage. Activation is enhanced by the binding of cAMP to cyclic nucleotide binding domains in each of the four subunits. In mammalians four isoforms of HCN channels have been identified (HCN1-4). The single-channel conductance of HCN channels has been described first in native cardiac channels (DiFrancesco, Nature, 1986). Its value was determined to be only ∼1 pS which is unusually small for a voltage gated cation channel. Surprisingly, in recombinant HCN2 channels a much larger conductance of ∼35 pS was determined (Michels et al., Circulation, 2005). Later Dekker and Yellen (J. Gen. Physiol., 2006) confirmed a small conductance of ∼1.5 pS but observed a pronounced cooperative gating of multiple channels. We expressed HCN2 channels in Xenopus oocytes and studied single channel currents in inside-out patches. Our results confirm a small conductance (∼2 pS) and do not provide any evidence for a cooperative gating between the channels, enabling recording from patches containing one and only one channel. The activating effect of cAMP, applied to the bath solution, is mediated by an increase of the open probability. In conclusion, HCN2 channels expressed in Xenopus oocytes develop an only small single-channel conductance, gate as individual channels, and are activated by cAMP via an increase of the open probability. These results are of importance for modelling single-channel properties from macroscopic currents.

Cyclic nucleotide-gated (CNG) channels of olfactory neurons are heterotetramers composed of three homologue subunits, 2xCNGA2, CNGA4 and CNGB1b. CNGA4 and CNGB1b subunits contribute to functional channels only in heterotetramers with CNGA2 subunits but not when expressed alone. It is well established that cAMP and cGMP can activate homotetrameric CNGA2 channels by binding to the cyclic nucleotide-binding domain (CNBD) and that the presence of the CNGA4 and CNGB1b subunit in heterotetrameric channels leads to an increased apparent affinity for the cyclic nucleotides. Our main question is: Do the CNGA4 and CNGB1b contribute to channel opening by binding cyclic nucleotides or only through allosteric modulation? By expressing TFP-labelled CNGA4 and CNGB1b subunits in oocytes, whose membrane was counterstained from the extracellular side by fluorescently-labeled lectin (Alexa Fluor® 633 WGA), we show that the CNGA2 subunit significantly promotes the incorporation of the both the CNGA4 and the CNGB1b subunit within the plasma membrane of Xenopus oocytes. Activation of heterotetrameric CNGA2:A4:B1b channels was studied in excised patches by measuring simultaneously ligand binding and activation gating by using a fluorescent cGMP derivative. To study the ligand binding to the CNGA4 and CNGB1b subunit only, we performed experiments in channels with specifically disabled CNBDs. We show that cGMP activates heterotetrameric olfactory channels by binding to the CNGA2 and CNGA4 subunits only, whereas cAMP seems to activate the channels by binding to all three types of subunits. These findings suggest that the CNGB1b subunit plays an important role in distinguishing between cGMP and cAMP.

Cyclic nucleotide-gated (CNG) channels mediate signal transduction in photoreceptors and olfactory cells. Wild-type olfactory CNG channels are composed of three types of subunits, CNGA2, CNGA4, and CNGB1b. Out of these only CNGA2 subunits can form functional homotetrameric channels when expressed in heterologous systems. Homotetrameric CNGA2 channels are very useful for studying elementary biophysical processes. However, even for homotetrameric channels the transmission of the ligand binding to the pore opening is still a mystery. Undoubtedly clear is only that the activation of the channels is not generated by independently operating but by interacting subunits. We are interested in how a single subunit embedded in a channel binds a ligand and evokes channel activation, i.e. for the whole channel we intend to kinetically dissect the molecular gating mechanism induced by the first ligand binding step. We constructed tetrameric concatamers of CNGA2 channels with various numbers of wild-type and mutated binding domains of high and extremely low affinity. Ligand binding was measured by confocal patch-clamp fluorometry (frame rate up to 277 images per second) using a fluorescent cGMP analogue. Our results show that a single subunit operates a channel only at 23-fold higher ligand concentrations than a tetrameric channel composed of four cooperating subunits. Nevertheless, the single subunit opens the channel pore to the full conductance level. Moreover, we show that Markovian models with a discrete number of states surprisingly fail to describe the action of the only wild-type subunit. Instead, a model consisting of three separate but intimately coupled processes proved to be adequate for describing channel activation, containing a rapid ligand binding close to the diffusion limit, a continuous conformational diffusion upon gating consisting of two components, and a rapid and discrete pore action.

Cyclic nucleotide-gated (CNG) channels mediate sensory signal transduction in retinal and olfactory cells. The channels are activated by the binding of cyclic nucleotides to an intracellular cyclic nucleotide-binding domain (CNBD). The molecular events translating the binding to the pore opening are still unknown. We investigated the role of intracellular channel regions on the activation process by constructing chimeric channels in which the N-terminus, the S4-S5 linker, the C-terminus, and the CNBD of the retinal CNGA1 subunit were systematically replaced by respective regions of the olfactory CNGA2 subunit. Macroscopic concentration-response relations were analyzed, yielding the apparent affinity to cGMP and the Hill coefficient. The degree of functional coupling of intracellular regions in the activation gating was determined by an interaction energy according to the principles of thermodynamic double-mutant cycle analysis. We show that all four intracellular regions, including the short S4-S5 linker, are involved in controlling the apparent affinity of the channel to cGMP and, moreover, in determining the degree of cooperativity between the subunits as determined from the Hill coefficients. The interaction energies are specific for pairs of regions. The interaction energy of the S4-S5 region with both the N-terminus and the C-linker was significantly different from zero at all possible combinations of intracellular regions. In contrast, at all possible combinations of intracellular regions no significant interaction energy was observed for the S4-S5 linker and the CNBD. It is concluded that in CNG channels the S4-S5 linker cooperates with both N-terminus and C-linker in the process of translating ligand binding to the pore opening.